1. Field of the Invention
The present invention relates to a material for promoting the freezing of water or a hydrous substance, a carrier on which antifreeze proteins are immobilized, a method for promoting the freezing of water or a hydrous substance, a method for freezing water or a hydrous substance while removing gases contained therein, and a method for freezing a water component in a hydrous substance while concentrating components other than water in the hydrous substance.
2. Background Art
It is generally thought that water freezes at 0° C. However, water does not freeze at or lower than 0° C. (supercool state), unless a trigger substance for freezing, that is, an “ice nucleus,” is present. To naturally cause the generation of ice nuclei within water or a hydrous substance in a supercool state and thus cause the freezing thereof, such a substance must be cooled to a low temperature between −20° C. (general freezer temperature) and −196° C. (liquid nitrogen temperature), for example. Meanwhile, when ice nuclei are caused to be present, water or a hydrous substance can be frozen at a high temperature of 0° C. or lower. Examples of known ice nucleus substances, that is, substances that promote freezing, include silver iodide and ice nucleation-active bacteria, which enable the freezing of water at −8° C. and temperatures between −4 and −2° C., respectively. In particular, large amounts of cooling energy consumption is a problem in technical fields including the manufacturing industry, the processing industry, the freezing industry, the ice-making industry, the thermal preservation industry, food preservation, cell preservation, organ preservation, rainmaking, and artificial snowfall. Hence, the use of ice nucleus substances in the relevant technical fields has been expected. Specifically, the freezing of water with low energy (at high temperatures of 0° C. or lower) through the use of ice nucleus substances has been expected. However, the use of the above-described ice nucleus substances results in the presence of a mixture of heavy metals and bacteria in a hydrous substance. There is concern that this adversely affects the environment, human bodies, the ecosystem, and the like. Furthermore, applicable fields and ranges are extremely limited because of cost (ice nucleation-active bacteria are relatively expensive) and the like. Therefore, development of a new material that can function as an ice nucleus and can exert activity to promote the freezing of water has been strongly desired.
Moreover, when water is frozen by a general method, cloudy (milky) ice is generally formed because of the effects of air and the like dissolved in water. In the meantime, regarding ice to be used for beverages (e.g., beverages with water, beverages “on the rocks,” and cool drinks), food preservation, decorative ice cylinders, or the like, ice with a low impurity level and high clarity is thought to be suitable because of its difficulty in thawing, its high beauty, and its ability to evoke a refreshingly cool feeling. Because of a rising trend toward cleanliness in recent years, ice-making apparatuses or the like with which clear ice can be produced are increasingly incorporated not only in ice-makers for business use but also in household refrigerators. Conventional clear ice production methods are: a method that involves freezing by spraying hot air from the top surface of an ice tray at a predetermined angle; a method that involves freezing in turn starting from the lower surface of an ice tray using a Peltier element; a method that involves supplying water to an ice tray and then vibrating the ice tray; a method that involves degassing dissolved gases such as air in water before ice making; a method that involves lowering an opening of an ice tray, supplying water in the form of a fountain, and then gradually freezing an ice making container (from the side thereof to the whole container); and a method that involves sending cool air to the back of an ice tray and then freezing while heating using a heater embedded in the cover of the ice tray, and the like. Specifically, temperature control for water to be frozen or an ice-making container, complicated tasks such as degassing or extrusion of water, and apparatuses and related costs have been required for clear ice production. Furthermore, large quantities of cooling energy at around −20° C. have been required for freezing. Hence, development of a new method for conveniently producing clear ice at a low cost and with low energy has been strongly desired.
Furthermore, an example of technology using water freezing is freeze concentration technology. This technology is a method that involves partially freezing a hydrous substance to separate water in the form of ice, so as to concentrate a solute. The technology has major advantages over other concentration methods, such that it enables uniform concentration of a solute component and has a high ability to preserve the qualities of a solute. It is expected to apply the freeze concentration technology to concentration (e.g., for liquid foods, medicines, or liquid fuel) and to clarification treatment or wastewater treatment (e.g., for waste water or waste liquid). Various apparatuses for such applications have been developed. However, all of these apparatuses have mechanisms by which a liquid to be treated is frozen by supercooling. Hence, the apparatuses are problematic in that solutes are easily incorporated in ice, the resulting concentration rates are low, and the energy efficiencies are poor. The apparatuses are also problematic in that a concentration unit with multiple stages must be provided because of the low concentration rate of a concentration unit with a single stage. This results in higher priced, more complicated, and larger apparatuses. Moreover, a freezing machine has been required for every concentration unit, so that large quantities of energy have been required for obtaining frozen products. Accordingly, development of a new technique capable of conveniently freezing and concentrating a target at low cost with low energy but without requiring supercooling has been strongly desired.
An antifreeze protein is relatively a small protein comprising a polypeptide of approximately 30 to 150 residues. It is known that an antifreeze protein is capable of inhibiting the freezing of water or a hydrous substance by binding to a specific crystal surface of an ice nucleus so as to suppress the ice crystal growth within a temperature range of 0° C. or lower. In recent years, the structures of various antifreeze proteins have been revealed by an NMR method or an X-ray crystal structure analysis method, so that their sites for binding to ice crystals have been revealed (Davies P. L. and Sykes B. D. 1997. Curr. Opin. Struct. Biol. 7: 828-834).
In the meantime, an ice nucleation protein is a protein that promotes the ice crystal growth. For example, an ice nucleation protein derived from Pseudomonas syringae, which is a kind of ice nucleation bacteria, is a membrane protein comprising approximately 1200 amino acids and being composed of 3 regions: an N-terminal region (up to 19 kDa); a central region (up to 94 kDa); and a C-terminal region (up to 7 kDa). Of these 3 regions, the central region is composed of a characteristic repeating amino acid sequence, and the sequence is thought to be a site that exerts ice nucleation activity (Hew C. L. and Yang D. S. C., 1992. Eur. J. Biochem. 203: 33-42).
An antifreeze protein and an ice nucleation protein share no homology in terms of amino acid sequence. Furthermore, the molecular weight of an ice nucleation protein is 10 to 20 or more times larger than that of an antifreeze protein.
In general, an antifreeze protein is stable and can be easily extracted from organisms containing the protein (JP Patent Publication (Kokai) No. 2004-344033 A) or easily obtained using a genetic engineering technique. On the other hand, no technology for stably isolating and purifying an ice nucleation protein in a large amount has been developed. Hence, it is extremely difficult to apply a pure ice nucleation protein as a material for promoting the freezing of water.